Human γS-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of ßγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein's tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

The Y46D Mutation Destabilizes Dense Packing of the Second Greek Key Pair of Human γC-Crystallin Causing Congenital Nuclear Cataracts.

The γ-crystallins are highly expressed structural lens proteins comprising four Greek key motifs arranged in two domains. Their globular structure and short-range spatial ordering are essential for lens transparency. Aromatic residues play a vital role in stabilizing Greek key folds by forming Greek key or non-Greek key pairs or tyrosine corners. We investigated the effects of the cataractogenic Y46D mutation in the second Greek key pair (Y46-Y51) of human γC-crystallin on its stability and aggregation. Wild-type and Y46D mutant human γC-crystallin were overexpressed in E. coli BL-21(DE3) PLysS cells, purified using ion-exchange and size-exclusion chromatography, and analyzed by fluorescence spectroscopy and circular dichroism spectroscopy. The Y46D mutation does not affect the γC-crystallin backbone conformation under benign conditions but alters the tryptophan microenvironment, exposing hydrophobic residues to the surface. The Y46D mutant undergoes a three-state transition under thermal stress with midpoints of 54.6 and 67.7 °C while the wild type shows a two-state transition with a midpoint of 77.6 °C. The Y46D mutant also shows a three-state transition under GuHCl stress with Cm values of 0.9 and 2.1 M while the wild type shows a two-state transition with a Cm of 2.4 M GuHCl. Mutant but not wild-type γC-crystallin forms light scattering particles upon heating at 65 °C. Overall, the Y46D CRYGS mutation leaves the protein fold intact under benign conditions but destabilizes the molecule by altering the tryptophan microenvironment and exposing hydrophobic residues to its surface, thus increasing its susceptibility to thermal and chemical stress with resultant self-aggregation, light scattering, and cataract.

Impact of hydrogen peroxide on structure, stability, and aggregational properties of human γS-crystallin.

Cataract is the leading cause of blindness worldwide. Oxidative stress is one of the known risk factors for agerelated cataracts. The present study was designed to understand the effect of H2O2-induced oxidative stress on human γS-crystallin and its relationship to lens opacification and cataract. Human γS-crystallin cDNA was cloned into the pET-20b vector, overexpressed in BL21 Star (DE3) cells, and was purified using ion-exchange and gel filtration chromatography. The structure, stability, and aggregational properties of human γS-crystallin under H2O2 stress were studied using fluorescence and circular dichroism spectroscopy methods. H2O2 treatment did not show any significant effect on the γS-crystallin secondary structure but showed an effect on its tertiary structure, resulting in N'-formylkynurenine formation. The H2O2-treated sample showed increased surface hydrophobicity, was less stable, and opened its Greek key motifs earlier with a midpoint of thermal unfolding curve (Tm) of 70.2°C compared with untreated γS-crystallin (Tm=71.4°C). The sample treated with H2O2 aggregated earlier in response to heating at 65°C. H2O2-induced oxidative stress alters the tryptophan microenvironment and the surface hydrophobicity of γS-crystallin, and these changes decrease its thermal stability and increase its tendency to aggregate, consistent with its role as a risk factor in age-related cataract.

Gamma crystallins of the human eye lens.

BACKGROUND: Protein crystallins co me in three types (α, ß and γ) and are found predominantly in the eye, and particularly in the lens, where they are packed into a compact, plastic, elastic, and transparent globule of proper refractive power range that aids in focusing incoming light on to the retina. Of these, the γ-crystallins are found largely in the nuclear region of the lens at very high concentrations (>400 mg/ml). The connection between their structure and inter-molecular interactions and lens transparency is an issue of particular interest. SCOPE OF REVIEW: We review the origin and phylogeny of the gamma crystallins, their special structure involving the use of Greek key supersecondary structural motif, and how they aid in offering the appropriate refractive index gradient, intermolecular short range attractive interactions (aiding in packing them into a transparent ball), the role that several of the constituent amino acid residues play in this process, the thermodynamic and kinetic stability and how even single point mutations can upset this delicate balance and lead to intermolecular aggregation, forming light-scattering particles which compromise transparency. We cite several examples of this, and illustrate this by cloning, expressing, isolating and comparing the properties of the mutant protein S39C of human γS-crystallin (associated with congenital cataract-microcornea), with those of the wild type molecule. In addition, we note that human γ-crystallins are also present in other parts of the eye (e.g., retina), where their functions are yet to be understood. MAJOR CONCLUSIONS: There are several 'crucial' residues in and around the Greek key motifs which are essential to maintain the compact architecture of the crystallin molecules. We find that a mutation that replaces even one of these residues can lead to reduction in solubility, formation of light-scattering particles and loss of transparency in the molecular assembly. GENERAL SIGNIFICANCE: Such a molecular understanding of the process helps us construct the continuum of genotype-molecular structural phenotype-clinical (pathological) phenotype. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Mutations in the Corneal Endothelial Dystrophy-Associated Gene SLC4A11 Render the Cells More Vulnerable to Oxidative Insults.

PURPOSE: To investigate the effect of mutations in SLC4A11 on cellular localization of the protein, mitochondrial function, and apoptosis due to oxidative stress. Mutations in SLC4A11 have been associated with 2 different forms of corneal endothelial dystrophy that lead to degeneration of the corneal endothelium, causing opacity of the cornea and gradual vision loss. METHODS: HEK 293 cells were transfected with wild-type SLC4A11 or mutants, Ser213Leu, Arg233Cys, Gly418Asp, and Thr584Lys, and exposed to oxidative stress. Cellular localization of the proteins was detected by confocal microscopy, whereas mitochondrial dysfunction, reactive oxygen species (ROS) generation, and apoptosis were analyzed by flow cytometry and a colorimetric assay. Expressions of antioxidant genes were quantitated by real-time polymerase chain reaction. RESULTS: Although wild-type SLC4A11 was localized on the cell membrane, mutant proteins were found diffused in the cytoplasm. Mutations in SLC4A11 caused an increase in generation of ROS and mitochondrial dysfunction due to oxidative stress. NRF2, HO-1, and NQO expression decreased significantly, and a higher rate of apoptosis was detected in cells with mutant proteins under oxidative stress. CONCLUSIONS: Our data suggest that mutations in SLC4A11 cause retention of the protein in the cytoplasm and generate increased reactive oxygen species. We found that cells containing mutant SLC4A11 are more vulnerable to oxidative and mitochondrial damage, less able to overcome oxidative stress through the expression of sufficient levels of antioxidant genes, and are more prone to apoptotic death.

Structural integrity of the Greek key motif in ßγ-crystallins is vital for central eye lens transparency.

BACKGROUND: We highlight an unrecognized physiological role for the Greek key motif, an evolutionarily conserved super-secondary structural topology of the ßγ-crystallins. These proteins constitute the bulk of the human eye lens, packed at very high concentrations in a compact, globular, short-range order, generating transparency. Congenital cataract (affecting 400,000 newborns yearly worldwide), associated with 54 mutations in ßγ-crystallins, occurs in two major phenotypes nuclear cataract, which blocks the central visual axis, hampering the development of the growing eye and demanding earliest intervention, and the milder peripheral progressive cataract where surgery can wait. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations. METHODS: Wild type and several representative mutant proteins were cloned, expressed and purified and their secondary and tertiary structural details, as well as structural stability, were compared in solution, using spectroscopy. Their tendencies to aggregate in vitro and in cellulo were also compared. In addition, we analyzed their structural differences by molecular modeling in silico. RESULTS: Based on their properties, mutants are seen to fall into two classes. Mutants A36P, L45PL54P, R140X, and G165fs display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and disturb/distort the Greek key motif. And they are associated with nuclear cataract. In contrast, mutants P24T and R77S, associated with peripheral cataract, behave quite similar to the wild type molecule, and do not affect the Greek key topology. CONCLUSION: When a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display 'native state aggregation', leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy "distort motif, lose central vision".

Structural analysis of the mutant protein D26G of human γS-crystallin, associated with Coppock cataract.

PURPOSE: To analyze the protein structural features responsible for the aggregation properties of the mutant protein D26G human γS-crystallin (HGSC) associated with congenital Coppock-type cataract. METHODS: cDNAs of wild-type (WT) and D26G mutant HGSC were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. Their secondary and tertiary structural features, aggregation tendencies, and structural stabilities were compared using spectroscopic (circular dichroism, intrinsic and extrinsic fluorescence), molecular modeling, and dynamics methods. RESULTS: No difference was observed between the conformational (secondary and tertiary structural) features and aggregation properties between the WT and D26G proteins. The mutant, however, was structurally less stable; it denatured at a slightly lower concentration of the added chemical denaturant (at 2.05 M guanidinium chloride, cf. 2.20 M for the WT) and at a slightly lower temperature (at 70.8 °C, cf. 72.0 °C for the WT). The mutant also self-aggregated more readily (it turned turbid upon standing; at 65 °C, it started precipitating beyond 200 s, while the WT did not, even after 900 s). Molecular modeling showed that the Asp26-Arg84 contact (and the related Arg84-Asn54 interaction) was disturbed in the mutant, making the latter less compact around the mutation site. CONCLUSIONS: The cataract-associated mutant D26G of HGSC is remarkably close to the WT molecule in structural features, with only a microenvironmental change in the packing around the mutation site. This alteration appears sufficient to promote self-aggregation, resulting in peripheral cataract.

The mutation V42M distorts the compact packing of the human gamma-S-crystallin molecule, resulting in congenital cataract.

BACKGROUND: Human γS-crystallin is an important component of the human eye lens nucleus and cortex. The mutation V42M in the molecule causes severe congenital cataract in children. We compare the structure of the mutant protein with that of the wild type in order to understand how structural changes in the mutant relate to the mechanism of opacification. METHODS: Both proteins were made using conventional cloning and expression procedures. Secondary and tertiary structural features of the proteins were analyzed using spectral methods. Structural stabilities of the proteins were analyzed using chemical and thermal denaturation methods. Self-aggregation was monitored using extrinsic spectral probes. Molecular modeling was used to compare the structural features of the two proteins. RESULTS: While the wild type and mutant have the same secondary structure, molecular modeling and fluorescence analysis suggest the mutant to have a more open tertiary structure, with a larger hydrophobic surface. Experiments using extrinsic probes reveal that the mutant readily self-aggregates, with the suggestion that the aggregates might be similar to amyloidogenic fibrils. Chemical denaturation indicates that while the wild type exhibits the classic two-state transition, V42M goes through an intermediate state, and has a distinctly lower stability than the wild type. The temperature of thermal unfolding of the mutant is also distinctly lower. Further, the mutant readily precipitates and scatters light more easily than the wild type. CONCLUSION: The replacement of valine in position 42 by the longer and bulkier methionine in human γS-crystallin perturbs the compact ß-sheet core packing topology in the N-terminal domain of the molecule, exposes nonpolar residues thereby increasing the surface hydrophobicity and weakens the stability of the protein, thus promoting self-aggregation leading to light scattering particles. This set of changes in the properties of the mutant offers a molecular insight into the mechanism of opacification.

Structural and aggregation behavior of the human γD-crystallin mutant E107A, associated with congenital nuclear cataract.

PURPOSE: To analyze the conformational features and aggregation properties of the mutant protein E107A human γD-crystallin (HGDC), associated with congenital nuclear cataract. METHODS: cDNAs of wild type and E107A mutant were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. The conformational properties and structural stability of the two proteins were compared using circular dichroism and fluorescence spectroscopic analysis. His-tagged cDNAs of the two proteins were transfected into HLE-3B human lens epithelial cells, and into HeLa cells and their in situ aggregation properties compared using immunofluorescence. RESULTS: The mutant protein was found to be remarkably similar in its secondary and tertiary structural features to the wild type. Its structural stability, analyzed by guanidinium chloride-induced denaturation, was also found to be similar. Its solubility, however, was over hundred-fold less than that of the wild type, and it had the tendency to precipitate and form light scattering particles. That it had the tendency to self- aggregate was noticed by using bis-ANS and Nile Red as extrinsic fluorescent probes. Such aggregation was also seen in situ when transfected and expressed in HLE-3B and in HeLa cell lines. CONCLUSIONS: E107A HGDC is yet another example of how a point mutation in the protein does not affect its conformation and stability but leads to substantial reduction in solubility and generation of light scattering aggregate particles in vitro and in situ when introduced into cell lines.